A lipid analogue that inhibits sphingomyelin hydrolysis and synthesis, increases ceramide, and leads to cell death

Abstract

We report the synthesis and characterization of a novel thiourea derivative of sphingomyelin (AD2765). In vitro assays using pure enzyme and/or cell extracts revealed that this compound inhibited the hydrolysis of BODIPY-conjugated or 14C-labeled sphingomyelin by acid sphingomyelinase and Mg2+-dependent neutral sphingomyelinase. Studies in normal human skin fibroblasts further revealed that AD2765 was taken up by cells and inhibited the hydrolysis of BODIPY-conjugated sphingomyelin in situ. In situ and in vitro studies also showed that this compound inhibited the synthesis of sphingomyelin from BODIPY-conjugated ceramide. The specificity of AD2765 for enzymes involved in sphingomyelin metabolism was demonstrated by the fact that it had no effect on the hydrolysis of BODIPY-conjugated ceramide by acid ceramidase or on the synthesis of BODIPY-conjugated glucosylceramide from BODIPY-conjugated ceramide. The overall effect of AD2765 on sphingomyelin metabolism was concentration-dependent, and treatment of normal human skin fibroblasts or cancer cells with this compound at concentrations > 10 μM led to an increase in cellular ceramide and cell death.Thus, AD2765 might be used to manipulate sphingomyelin metabolism in various ways, potentially to reduce substrate accumulation in cells from types A and B Niemann-Pick disease patients, and/or to affect the growth of human cancer cells. We report the synthesis and characterization of a novel thiourea derivative of sphingomyelin (AD2765). In vitro assays using pure enzyme and/or cell extracts revealed that this compound inhibited the hydrolysis of BODIPY-conjugated or 14C-labeled sphingomyelin by acid sphingomyelinase and Mg2+-dependent neutral sphingomyelinase. Studies in normal human skin fibroblasts further revealed that AD2765 was taken up by cells and inhibited the hydrolysis of BODIPY-conjugated sphingomyelin in situ. In situ and in vitro studies also showed that this compound inhibited the synthesis of sphingomyelin from BODIPY-conjugated ceramide. The specificity of AD2765 for enzymes involved in sphingomyelin metabolism was demonstrated by the fact that it had no effect on the hydrolysis of BODIPY-conjugated ceramide by acid ceramidase or on the synthesis of BODIPY-conjugated glucosylceramide from BODIPY-conjugated ceramide. The overall effect of AD2765 on sphingomyelin metabolism was concentration-dependent, and treatment of normal human skin fibroblasts or cancer cells with this compound at concentrations > 10 μM led to an increase in cellular ceramide and cell death. Thus, AD2765 might be used to manipulate sphingomyelin metabolism in various ways, potentially to reduce substrate accumulation in cells from types A and B Niemann-Pick disease patients, and/or to affect the growth of human cancer cells. Sphingomyelin is an important component of cell membranes and a major source of ceramide involved in signal transduction (1Levade T. Jaffrezou J.P. Signalling sphingomyelinases: which, where, how and why?.Biochim. Biophys. Acta. 1999; 1438: 1-17Crossref PubMed Scopus (283) Google Scholar). Human acid sphingomyelinase (ASM; sphingomyelin phosphodiesterase, EC 3.1.4.12) is one enzyme that catalyzes the hydrolysis of sphingomyelin, resulting in the formation of ceramide and phosphocholine (2Barenholz Y. Roitman R. Gatt S. Enzymatic hydrolysis of sphingolipids. II. Hydrolysis of sphingomyelin by an enzyme from rat brain.J. Biol. Chem. 1966; 241: 3731-3737PubMed Google Scholar). ASM is found predominantly within lysosomes, but other enzyme forms, including a secreted, Zn2+-dependant form (3Tabas I. Secretory sphingomyelinase.Chem. Phys. Lipids. 1999; 102: 123-130Crossref PubMed Scopus (96) Google Scholar) and a plasma membrane form localized to raft structures (4Liu P. Anderson R.G. 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Chem. 1991; 266: 8531-8539Abstract Full Text PDF PubMed Google Scholar), leading to the production of recombinant enzyme in overexpressing CHO cells (12He X. Miranda S.R. Xiong X. Dagan A. Gatt S. Schuchman E.H. Characterization of human acid sphingomyelinase purified from the media of overexpressing Chinese hamster ovary cells.Biochim. Biophys. Acta. 1999; 1432: 251-264Crossref PubMed Scopus (84) Google Scholar) and insect cells (13Bartelsen O. Lansmann S. Nettersheim M. Lemm T. Ferlinz K. Sandhoff K. Expression of recombinant human acid sphingomyelinase in insect Sf21 cells: purification, processing and enzymatic characterization.J. Biotechnol. 1998; 63: 29-40Crossref PubMed Scopus (30) Google Scholar). Other neutral and alkaline sphingomyelinase activities also exist in mammalian cells, but these are distinct enzymes derived from different genes (1Levade T. Jaffrezou J.P. Signalling sphingomyelinases: which, where, how and why?.Biochim. Biophys. Acta. 1999; 1438: 1-17Crossref PubMed Scopus (283) Google Scholar). In humans, inherited mutations in the SMPD-1 gene result in types A and B Niemann-Pick disease (NPD) (14Schuchman E.H. Desnick R.J. Niemann-Pick disease type A and B: acid-sphingomyelinase deficiencies.in: Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Basis of Inherited Disease. 8th edition. McGraw-Hill, New York2001: 2601-2624Google Scholar). In addition, a recent report showed that deletion of the gene (Smpd3) encoding one of the neutral sphingomyelinases in mice results in osteogenesis and dentiogenesis imperfecta (15Aubin I. Adams C.P. Opsahl S. Septier D. Bishop C.E. Auge N. Salvayre R. Begre-Salvayre A. Goldberg M. Guenet J.L. et al.A deletion in the gene encoding sphingomyelin phosphodiesterase 3 (Smpd3) results in osteogenesis and dentinogenesis imperfecta in the mouse.Nat. Genet. 2005; 37: 803-805Crossref PubMed Scopus (138) Google Scholar). As noted above, the reaction product of ASM activity, ceramide, is a potent lipid second messenger involved in diverse cellular functions, including cell growth, differentiation, and death (16Ruvolo P.P. Intracellular signal transduction pathways activated by ceramide and its metabolites.Pharmacol. Res. 2003; 47: 383-392Crossref PubMed Scopus (295) Google Scholar, 17Gulbins E. Kolesnick R. Raft ceramide in molecular medicine.Oncogene. 2003; 22: 7070-7077Crossref PubMed Scopus (351) Google Scholar). Ceramide production has been implicated in the pathogenesis of diseases such as atherosclerosis and acquired immunodeficiency syndrome, as well as in liver damage and acute lung injury (18Barnes P.J. Ceramide lances the lungs.Nat. Med. 2004; 10: 130-131Crossref PubMed Scopus (18) Google Scholar, 19Mari M. Colell A. Morales A. Paneda C. Varela-Nieto I. Garcia-Ruiz C. Fernandez-Checa J.C. Acidic sphingomyelinase downregulates the liver-specific methionine adenosyltransferase 1A, contributing to tumor necrosis factor-induced lethal hepatitis.J. Clin. Invest. 2004; 113: 895-904Crossref PubMed Scopus (71) Google Scholar). The need for greater understanding of sphingomyelin and ceramide metabolism, as well as the potential clinical significance of modulating this pathway, has prompted the development of several classes of sphingomyelinase inhibitors. For example, various synthetic sphingomyelin analogues (20Yokomatsu T. Murano T. Akiyama T. Koizumi J. Shibuya S. Tsuji Y. Soeda S. Shimeno H. Synthesis of non-competitive inhibitors of sphingomyelinases with significant activity.Bioorg. Med. Chem. Lett. 2003; 13: 229-236Crossref PubMed Scopus (66) Google Scholar, 21Hakogi T. Monden Y. Taichi M. Iwama S. Fujii S. Ikeda K. Katsumura S. Synthesis of sphingomyelin carbon analogues as sphingomyelinase inhibitors.J. Org. Chem. 2002; 67: 4839-4846Crossref PubMed Scopus (44) Google Scholar, 22Taguchi M. Goda K. Sugimoto K. Akama T. Yamamoto K. Suzuki T. Tomishima Y. Nishiguchi M. Arai K. Takahashi K. et al.Biological evaluation of sphingomyelin analogues as inhibitors of sphingomyelinase.Bioorg. Med. Chem. Lett. 2003; 13: 3681-3684Crossref PubMed Scopus (25) Google Scholar), as well as analogues of naturally occurring inhibitors, such as α-mangostin, a natural product derived from Garcinia mangostana, have been developed (23Hamada M. Iikubo K. Ishikawa Y. Ikeda A. Umezawa K. Nishiyama S. Biological activities of alpha-mangostin derivatives against acidic sphingomyelinase.Bioorg. Med. Chem. Lett. 2003; 13: 3151-3153Crossref PubMed Scopus (33) Google Scholar). In addition, the lipid phosphatidylinositol-3,5-bisphosphate has been shown to inhibit ASM activity (24Kolzer M. Arenz C. Ferlinz K. Werth N. Schulze H. R. Sandhoff K. is a potent and of acid Chem. 2003; PubMed Scopus Google Scholar). sphingomyelinase inhibitors be for a of For example, sphingomyelinase inhibitors potentially be used to the growth of cancer cells, to the of in the of ASM inhibitors, as molecular for the treatment of types A and B R.J. Schuchman E.H. and from Genet. 2002; PubMed Scopus Google Scholar, J.W. to 2003; PubMed Scopus Google Scholar). was by the of and for the treatment of disease S. N. Suzuki Y. and of A in by an enzyme Med. 1999; PubMed Scopus Google Scholar, N. S. H. Ikeda K. K. A. In vitro and of A activity in by and its J. PubMed Scopus Google Scholar). sphingomyelinase inhibitors are the these have on sphingomyelin metabolism in cells have developed a sphingomyelin and it in vitro and in cells. We that this compound has on sphingomyelin metabolism, including the of ASM and Mg2+-dependent neutral sphingomyelinase as well as on the of sphingomyelin no on ceramide hydrolysis or on the synthesis of In addition, treatment of cells with AD2765 at concentrations > 10 μM led to an increase in cellular ceramide and cell death. The potential of this compound are Human skin fibroblasts in with and Human and cells in with and cells in with and cells in with and of was in of in a 10 the was of The was to for and to a and of of and of The was and the to The was with of and to The product was purified by using for by an and with in a The of product from this reaction was and the as by was A of AD2765 was as that was used of the The product was ASM activity was in vitro using and sphingomyelin assays as X. Dagan A. Gatt S. Schuchman E.H. A to acid sphingomyelinase activity and types A and B Niemann-Pick 2003; PubMed Scopus Google Scholar). of cell extracts by of in 10 or purified enzyme with μM Molecular in and The reaction at for and by the of and the product was and by analysis using a The was as with the was with of and was used in the in of The reaction was a at by the of of and of and for at to a The the reaction product was for with an The activity of Mg2+-dependent neutral sphingomyelinase was in vitro using the for the ASM that the used and Acid ceramidase activity was in vitro as X. C.M. Dagan A. Gatt S. Schuchman E.H. A to acid ceramidase 1999; PubMed Scopus Google Scholar). the 10 reaction of of enzyme source by of in of μM and at for The reaction was by the of and the acid product was and by analysis using a or cells in of the and The in for and in a for 3 and was For the of concentrations of the compound in and the for at or ceramide a in was and at was for of was to the the and and the was The was a of and the was in of and to a of or sphingomyelin and or glucosylceramide also The was developed in and the of the sphingomyelin and glucosylceramide was using a with the using as a of human skin fibroblasts was as S. Miranda S.R. J.W. Dagan A. Gatt S. Schuchman E.H. of and cells from acid for Niemann-Pick disease gene and the development of cell gene 1999; PubMed Google Scholar). was by with at a of The was and the lipid was in by Human skin fibroblasts with of at for was by and of the with growth The cells for a further the fibroblasts by and at and the in of cells taken for and the was processed as at and the cell was in of The resulting cell was for by at for 10 and a further The was and the lipid was in of The was and by analysis using a as for the ASM activity sphingomyelin and glucosylceramide synthesis in the by Dagan et A. C. E. Gatt S. inhibit the of ceramide and cell Biophys. Acta. 2003; PubMed Scopus Google Scholar) A of cells was with or AD2765 for in was and the for an 3 by and with the cell was of and and the was of was to the was and and the was a of The was in and to a The was developed in and the of the and glucosylceramide was by with and using a The ceramide in cells was by a X. Dagan A. Gatt S. Schuchman E.H. analysis of ceramide and in by hydrolysis with 2005; PubMed Scopus Google Scholar). a lipid was using and in the The ceramide in these lipid extracts was using recombinant acid and the was with and by A of was to the of normal skin fibroblasts on at a of μM for in for at with in with and or processed further for membrane the of the the cells for at in normal and a with cells with for at at a of in normal and The for in by in for at with with at a of 10 for in by in as and and cell and a of a cell was in The resulting was to a and cells to the was as the of cells the A novel thiourea derivative of sphingomyelin and its to inhibit the hydrolysis of by recombinant human ASM was in As by the in this designated inhibited this reaction with an of also that these the of ASM was Studies using 14C-labeled sphingomyelin as the substrate the effect of AD2765 on the activity of pure ASM in vitro the of human recombinant acid sphingomyelinase activity by of purified ASM and concentrations of at different substrate concentrations from to In vitro assays as in and B: of ASM activity in cell human skin fibroblasts in the of AD2765 at the concentrations for and of ASM activity in of human cell and μM are as by We normal human skin fibroblasts with of AD2765 for the cells and used cell extracts to ASM As shown in significant of ASM activity was in the cell that AD2765 had the cells and was in the cells with μM AD2765 for had a of ASM activity, and the of this compound using the pure enzyme was μM this that AD2765 was in the cells the also to the of AD2765 on the activities of Mg2+-dependent neutral sphingomyelinase and acid As shown in the activity of Mg2+-dependent neutral sphingomyelinase but acid ceramidase was inhibited by this We to the of AD2765 to inhibit the hydrolysis of in situ. A was the cells with to the accumulation of the substrate within the cells, by a in the was to of the product in situ was by of and analysis using the X. Dagan A. Gatt S. Schuchman E.H. analysis of ceramide and in by hydrolysis with 2005; PubMed Scopus Google Scholar). As shown in 10 μM AD2765 was in the the of was the of this compound to cells and inhibit sphingomyelinase activity in situ. the the hydrolysis of and studies have that this lipid is to R. C. of to membrane the Phys. Lipids. 1999; 102: PubMed Scopus Google Scholar), the fact that AD2765 inhibit this reaction that this compound was at in to this this a derivative of sphingomyelin was and its and in cells by the derivative was to AD2765 the results revealed the of this compound and to and/or with the cell is important to that of the of the concentrations used for the is that at concentrations the compound might be to but this be further the of an in normal skin fibroblasts and cells with BODIPY-conjugated ceramide, and the to form sphingomyelin was as shown in this synthetic reaction also was inhibited 10 μM AD2765 was in the In concentrations of AD2765 inhibited the formation of sphingomyelin by this no on cell growth AD2765 was in the the same on sphingomyelin synthesis ceramide was used of ceramide these in situ the of AD2765 to inhibit the synthesis of sphingomyelin from ceramide in vitro was using extracts from cells. As shown in these results also showed that sphingomyelin synthesis was inhibited by cell extracts used to the in vitro on sphingomyelin as well as to the on the synthesis of As shown in AD2765 inhibited sphingomyelin synthesis from ceramide in cell but the synthesis of of normal human skin fibroblasts with μM AD2765 for led to a significant increase in ceramide, using 10 μM AD2765 potential source of this ceramide is of sphingomyelin as above, but it also was that AD2765 inhibit ceramidase activity, leading to a using pure acid ceramidase or cell extracts no of ceramidase activity by further the specificity of its for enzymes involved in sphingomyelin normal human skin human skin fibroblasts from a with and a human cell with AD2765 and the on cell at and As be this compound inhibited cell growth and led to cell death in of the cell in a 10 μM AD2765 significant cell death in these cells μM with a production of cellular ceramide AD2765 cell death in cells as well as in cells the cell death was on the of an signaling these results that cells in the of μM AD2765 for or the in situ effect was to be an increase of cellular ceramide and cell death. The hydrolysis of sphingomyelin by cellular sphingomyelinases to the production of ceramide, a potent lipid second messenger that apoptosis the of lipid raft structures (16Ruvolo P.P. Intracellular signal transduction pathways activated by ceramide and its metabolites.Pharmacol. Res. 2003; 47: 383-392Crossref PubMed Scopus (295) Google Scholar, 17Gulbins E. Kolesnick R. Raft ceramide in molecular medicine.Oncogene. 2003; 22: 7070-7077Crossref PubMed Scopus (351) Google Scholar, Y. of ceramide in cellular to PubMed Scopus Google Scholar). Ceramide is also the of major including other important second messenger such as and ceramide The of cell of the lipid Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). the of ceramide is to cell and the of disease, and this has led to the production of of modulating these such as inhibitors of sphingomyelinases and The of several sphingomyelinase inhibitors have been (20Yokomatsu T. Murano T. Akiyama T. Koizumi J. Shibuya S. Tsuji Y. Soeda S. Shimeno H. Synthesis of non-competitive inhibitors of sphingomyelinases with significant activity.Bioorg. Med. Chem. Lett. 2003; 13: 229-236Crossref PubMed Scopus (66) Google Scholar, 21Hakogi T. Monden Y. Taichi M. Iwama S. Fujii S. Ikeda K. Katsumura S. Synthesis of sphingomyelin carbon analogues as sphingomyelinase inhibitors.J. Org. Chem. 2002; 67: 4839-4846Crossref PubMed Scopus (44) Google Scholar, M. Iikubo K. Ishikawa Y. Ikeda A. Umezawa K. Nishiyama S. Biological activities of alpha-mangostin derivatives against acidic sphingomyelinase.Bioorg. Med. Chem. Lett. 2003; 13: 3151-3153Crossref PubMed Scopus (33) Google Scholar). inhibitors potentially be used to the growth of cancer cells, in the of ASM inhibitors, as molecular for the treatment of the synthesis and characterization of a novel sphingomyelin and that it inhibit the hydrolysis of in vitro and in cells. in vitro and in situ studies also revealed that AD2765 inhibit sphingomyelin synthesis as these specific for enzymes involved in sphingomyelin metabolism, AD2765 had no effect on acid ceramidase activity or the synthesis of characterization of the of AD2765 on sphingomyelin hydrolysis involved analysis using recombinant human analysis showed that the of AD2765 was μM and that the these in vitro was We also the of AD2765 to inhibit the hydrolysis of in situ using a results the effect of this compound on sphingomyelin We also studies to the specificity of this As shown in AD2765 inhibited the activity of ASM in cell but also the activity of Mg2+-dependent neutral sphingomyelinase. was no effect on the activity of acid ceramidase using extracts or pure Thus, AD2765 to have a effect on sphingomyelin hydrolysis in vitro and in situ but no effect on ceramide the in situ studies inhibited in cells, skin fibroblasts of neutral sphingomyelinase activity with ASM and it has been shown that to S. Miranda S.R. J.W. Dagan A. Gatt S. Schuchman E.H. of and cells from acid for Niemann-Pick disease gene and the development of cell gene 1999; PubMed Google Scholar, R. C. of to membrane the Phys. Lipids. 1999; 102: PubMed Scopus Google Scholar), that of this in situ effect was to the of by using a derivative of on its the derivative is to have different with found it also inhibited the activity of recombinant human ASM in vitro and was to is important to that of the of this it was used at concentrations for the Thus, from these studies that the of is the it is that are to cells it be to the the on sphingomyelin synthesis in this In to the sphingomyelinase activity, it also was noted that concentrations of AD2765 inhibited cell growth and cell death in fibroblasts and human cells. of was found to this effect results in that that a sphingomyelinase such as this to ceramide a potential and growth In cells with μM AD2765 had an overall increase in ceramide We to the it is that AD2765 was by ASM within the leading to the production of a ceramide that using recombinant ASM in in vitro of AD2765 hydrolysis A second the from an important for in the of apoptosis J. T. G. K. of Chem. 2002; PubMed Google Scholar). For example, a recent report demonstrated the of apoptosis the of the of the T. K. G. J.C. of in cells apoptosis by of by Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). Thus, it is that AD2765 led to the of membranes and the of the AD2765 was a sphingomyelin it was that this compound inhibited sphingomyelin synthesis as well as leading to an overall increase in ceramide further the the of human fibroblasts derived from an with concentrations of the and it was found that it growth and cell death in a to that in the normal skin revealed that the We the of AD2765 to inhibit sphingomyelin synthesis using an in situ the of BODIPY-conjugated and ceramide within these inhibited in the of that this be a for the or in with the of in situ results in vitro using cell a on the enzymes involved in sphingomyelin an effect on ceramide a effect on sphingomyelin was no effect of AD2765 on the synthesis of glucosylceramide from BODIPY-conjugated ceramide, further the specificity of this compound for enzymes involved in sphingomyelin further is to the involved in the on cell and death. important is the to this compound in situ results on the of sphingomyelin hydrolysis and studies that the compound is at in to it is important to that these studies the of to other that to cells, a of compound to of sphingomyelin as well as and it is this that to ceramide accumulation and cell death. The of compound that to these is on the of compound the cells are as well as on how the compound is and treatment of cells with 10 μM AD2765 to ceramide or significant cell death the fact that this of compound inhibited sphingomyelin synthesis potential to these is that the of ceramide by 10 μM AD2765 is in other synthetic In the of ceramide by μM AD2765 these leading to an overall increase in cellular ceramide and cell death. of the effect was by that how the compound is to cells in with might affect it is and it in the that how or analogues are to cells affect and metabolism in cells T. Gatt S. A. Salvayre R. pathways of and of sphingomyelin by cells and the potential of the neutral Biol. Chem. 1991; Scholar, C. E. M. J. A. S. by cells: of cell with and are Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). In this the synthesis and characterization of a novel sphingomyelin that had on sphingomyelin metabolism in situ. The specificity of this compound for enzymes involved in sphingomyelin metabolism was demonstrated by the of effect on acid ceramidase activity and the synthesis of treatment of cells with μM AD2765 led to an increase in ceramide and cell death a that was of ASM AD2765 a compound for studies at modulating sphingomyelin and ceramide in cells and and have on how the compound is to cells and to it The L. of the of for on enzyme was by of and by a from the The of was in by from a of